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Antimicrobial Agents and Chemotherapy, February 2004, p. 589-595, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.589-595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine{dagger}

Cecile Le Saint,1 Raphael Terreux,2 Daniele Duval,3 Jacques Durant,4 Helene Ettesse,4 Pierre Dellamonica,4 Roger Guedj,3 Jean Pierre Vincent,1 and Anny Cupo1*

Institut de Pharmacologie Moleculaire et Cellulaire, Unite Mixte de Recherche CNRS 6097, Sophia Antipolis,1 Laboratoire de Chimie des Aromes-Syntheses-Interactions,2 Laboratoire de Chimie Bioorganique, Unite Mixte de Recherche CNRS 6001, Faculte des Sciences,3 Service des Maladies Infectieuses, Centre Hospitalier Universitaire l'Archet, Nice, France4

Received 28 April 2003/ Returned for modification 7 October 2003/ Accepted 14 October 2003

Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.

* Corresponding author. Mailing address: Institut de Pharmacologies Moleculaire et Cellulaire, Unite Mixte de Recherche CNRS 6097, Sophia Antipolis, 660 Route des Lucioles Valbonne, France. Phone: 33 493 95 77 63. Fax: 33 493 95 77 63. E-mail: cupo{at}ipmc.cnrs.fr.

{dagger} This work is dedicated to the memory of Elisa Cupo (1919-2003).

Antimicrobial Agents and Chemotherapy, February 2004, p. 589-595, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.589-595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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